Summary This proposal explores a new model of Fanconi Anemia (FA) pathogenesis based on our findings that FA proteins protect hematopoietic stem cells (HSCs) from replication stress during the rapid developmental expansion in the fetal liver (FL). FA is a recessively inherited DNA repair disorder with cancer predisposition and near uniform bone marrow (BM) failure. While most FA patients experience symptomatic failure in early school age, BM hematopoietic stem cell (HSC) numbers are already compromised much earlier in life. We recently reported that the physiologic onset of HSC deficits in FA knockout mice occurs in utero. We now show that deficits in FA first emerge in the FL, caused by replication stress-associated Atr/Chk1 checkpoint engagement in immunophenotypically defined HSC. Because the HSC pool is typically complete at birth, these constraints constitute not only a previously unrecognized bottleneck for HSC pool formation in FA, but also pose a principal risk factor for rapid postnatal HSC exhaustion. To understand the exaggerated developmental vulnerability in the FA FL we have conducted additional preliminary studies of the FL microenvironment that implicate a subset of supportive cells forming the HSC niche. Altogether, we hypothesize that the physiological role of FA proteins is to safeguard in HSC pool clonality and genome integrity under conditions of replication stress. These observations lead us to test the long-term impact of fetal deficits in FA on HSC self-renewal and hematopoietic reserve. Specific Aim 1 Determine absolute HSC pool size and clonal diversity as driving risk factors for HSC exhaustion in FA Specific Aim 2 Dissect the long-term impact of checkpoint activation on genome stability and function in fetal FA HSC Specific Aim 3 Identify the FL specific niche abnormalities that contribute to hematopoietic deficits in FA, and reveal the key signaling pathways that functionally limit HSC expansion Altogether, this project advances a new paradigm, whereby FA proteins enable developmental expansion and self-renewal divisions critical to clonal diversity and genome stability in the HSC pool. This positions developmental deficits in FA patients as a driving risk factor for HSC exhaustion and a critical cause for morbidity and mortality. Results will provide insight for the development of safe and effective new therapies that mitigate loss of hematopoietic function in FA.